Resuscitation-Promoting Factors Are Cell Wall-Lytic Enzymes with Important Roles in the Germination and Growth of Streptomyces coelicolor

Dormancy is a common strategy adopted by bacterial cells as a means of surviving adverse environmental conditions. For Streptomyces bacteria, this involves developing chains of dormant exospores that extend away from the colony surface. Both spore formation and subsequent spore germination are tightly controlled processes, and while significant progress has been made in understanding the underlying regulatory and enzymatic bases for these, there are still significant gaps in our understanding. One class of proteins with a potential role in spore-associated processes are the so-called resuscitation-promoting factors, or Rpfs, which in other actinobacteria are needed to restore active growth to dormant cell populations. The model species Streptomyces coelicolor encodes five Rpf proteins (RpfA to RfpE), and here we show that these proteins have overlapping functions during growth. Collectively, the S. coelicolor Rpfs promote spore germination and are critical for growth under nutrient-limiting conditions. Previous studies have revealed structural similarities between the Rpf domain and lysozyme, and our in vitro biochemical assays revealed various levels of peptidoglycan cleavage capabilities for each of these five Streptomyces enzymes. Peptidoglycan remodeling by enzymes such as these must be stringently governed so as to retain the structural integrity of the cell wall. Our results suggest that one of the Rpfs, RpfB, is subject to a unique mode of enzymatic autoregulation, mediated by a domain of previously unknown function (DUF348) located within the N terminus of the protein; removal of this domain led to significantly enhanced peptidoglycan cleavage.     

Two novel Streptomyces protein protease inhibitors. Purification, activity, cloning, and expression.

In contrast to the Gram-negative bacteria, Gram-positive bacteria such as Streptomyces lack a mucopolysaccharide cell wall which allows them to produce and secrete a variety of proteins directly into their environment. In an effort to understand and eventually exploit the synthesis and secretion of proteins by Streptomyces, we identified and characterized two naturally occurring abundantly produced proteins in culture supernatants of Streptomyces lividans and Streptomyces longisporus. We purified these 10-kDa proteins and obtained partial amino acid sequence information which was then used to design oligonucleotide probes in order to clone their genes. Analysis of the sequence data indicated that these proteins were related to each other and to several other previously characterized Streptomyces protein protease inhibitors. We demonstrate that both proteins are protein protease inhibitors with specificity for trypsin-like enzymes. The presumptive signal peptidase cleavage sites and subsequent aminopeptidase products of each protein are characterized. Finally, we show that the cloned genes contain all of the information necessary to direct synthesis and secretion of the proteins by Streptomyces spp. or Escherichia coli.     

PlyC, a novel bacteriophage lysin for compartment-dependent proteomics of group A streptococci

Streptococcus pyogenes (Spy) (group A streptococci) is an important and exclusively human bacterial pathogen, which uses secreted and surface-associated proteins to circumvent the innate host defense mechanisms and to adhere and internalize into host cells. Thus, investigation of the bacterial extracellular compartments, including secreted and cell wall-associated subproteomes, is crucial for understanding adherence, invasion, and internalization mechanisms as major steps of Spy pathogenesis. Here, we compared a bacteriophage encoded cell wall hydrolase, PlyC, a multimeric lysin of the C1 bacteriophage, with the established glycosidase, mutanolysin, from Streptomyces globisporus for their suitability to efficiently digest Spy cell walls and release cell wall-anchored Spy proteins for subsequent proteome research. Our results show that PlyC is superior for cell wall protein extraction compared to mutanolysin due to its higher activity and specificity as an N-acetylmuramoyl-L-alanine amidase. Furthermore, our experimental design allowed us to delineate the actual localization of the proteins despite contamination with intracellular proteins.     

Induction of programmed lysis in <Emphasis Type="Italic">Streptomyces lividans</Emphasis> culture by the inhibitors of eukaryotic type serine/threonine protein kinases

Programmed death (PD) of the mycelium of Streptomyces lividans, namely, its delayed lysis in response to treatment with indolylmaleimide derivatives, which inhibit actinobacterial serine/threonine protein kinases (STPK), is described. Delayed lysis of mycelial cell was accompanied by DNA damage similar to PD in differentiating S. lividans mycelium. Two-dimensional electrophoresis and mass spectrometry were used to identify proteins up-regulated by a PD-inducing STPK inhibitor. Most of these proteins are known to be implicated in responses to various stress stimuli. Thus, our model of delayed cell lysis of actinobacteria upon STPK inhibition may serve for unveiling the molecular mechanisms of bacterial PD and for antimicrobial drug design.     

Pleiotropic functions of a Streptomyces pristinaespiralis autoregulator receptor in development, antibiotic biosynthesis, and expression of a superoxide dismutase

In Streptomyces, a family of related butyrolactones and their corresponding receptor proteins serve as quorum-sensing systems that can activate morphological development and antibiotic biosynthesis. Streptomyces pristinaespiralis contains a gene cluster encoding enzymes and regulatory proteins for the biosynthesis of pristinamycin, a clinically important streptogramin antibiotic complex. One of these proteins, PapR1, belongs to a well known family of Streptomyces antibiotic regulatory proteins. Gel shift assays using crude cytoplasmic extracts detected SpbR, a developmentally regulated protein that bound to the papR1 promoter. SpbR was purified, and its gene was cloned using reverse genetics. spbR encoded a 25-kDa protein similar to Streptomyces autoregulatory proteins of the butyrolactone receptor family, including scbR from Streptomyces coelicolor. In Escherichia coli, purified SpbR and ScbR produced bound sequences immediately upstream of papR1, spbR, and scbR. SpbR DNA-binding activity was inhibited by an extracellular metabolite with chromatographic properties similar to those of the well known gamma-butyrolactone signaling compounds. DNase I protection assays mapped the SpbR-binding site in the papR1 promoter to a sequence homologous to other known butyrolactone autoregulatory elements. A nucleotide data base search showed that these binding motifs were primarily located upstream of genes encoding Streptomyces antibiotic regulatory proteins and butyrolactone receptors in various Streptomyces species. Disruption of the spbR gene in S. pristinaespiralis resulted in severe defects in growth, morphological differentiation, pristinamycin biosynthesis, and expression of a secreted superoxide dismutase.     

The crystal structure of a penicilloyl-serine transferase of intermediate penicillin sensitivity. The DD-transpeptidase of streptomyces K15.

The serine DD-transpeptidase/penicillin-binding protein of Streptomyces K15 catalyzes peptide bond formation in a way that mimics the penicillin-sensitive peptide cross-linking reaction involved in bacterial cell wall peptidoglycan assembly. The Streptomyces K15 enzyme is peculiar in that it can be considered as an intermediate between classical penicillin-binding proteins, for which benzylpenicillin is a very efficient inactivator, and the resistant penicillin-binding proteins that have a low penicillin affinity. With its moderate penicillin sensitivity, the Streptomyces K15 DD-transpeptidase would be helpful in the understanding of the structure-activity relationship of this penicillin-recognizing protein superfamily. The structure of the Streptomyces K15 enzyme has been determined by x-ray crystallography at 2.0-A resolution and refined to an R-factor of 18.6%. The fold adopted by this 262-amino acid polypeptide generates a two-domain structure that is close to those of class A beta-lactamases. However, the Streptomyces K15 enzyme has two particular structural features. It lacks the amino-terminal alpha-helix found in the other penicilloyl-serine transferases, and it exhibits, at its surface, an additional four-stranded beta-sheet. These two characteristics might serve to anchor the enzyme in the plasma membrane. The overall topology of the catalytic pocket of the Streptomyces K15 enzyme is also comparable to that of the class A beta-lactamases, except that the Omega-loop, which bears the essential catalytic Glu(166) residue in the class A beta-lactamases, is entirely modified. This loop adopts a conformation similar to those found in the Streptomyces R61 DD-carboxypeptidase and class C beta-lactamases, with no equivalent acidic residue.     

A proteomic analysis of Streptomyces coelicolor programmed cell death

Programmed cell death (PCD) is an active cellular suicide that occurs in eukaryotes and bacteria in response to both abiotic and biotic stresses. In contrast to eukaryotic apoptosis, little is known about the molecular machinery that regulates bacterial PCD. In a previous work, we described the existence of PCD phenomena in Streptomyces (Manteca et al., Res. Microbiol. 2006, 157, 143-152). In the present study, we performed a proteomic analysis of PCD in Streptomyces coelicolor, for which we developed a system to obtain dead and live cell-enriched samples. PCD in this filamentous bacterium is accompanied by the appearance of enzymes involved in the degradation of cellular macromolecules, regulatory proteins, and stress-induced proteins. We argue that some of these proteins have specific functions in the PCD pathway and putative roles for the identified proteins have been proposed. The increased amounts of several antioxidant proteins suggest oxidative stress as either the cause or consequence of the cell death.     

Cell shape,division and development: the 2002 American Society for Microbiology (ASM) conference on prokaryotic development

In the last decade, the use of cytological techniques, together with the analysis of complete genomes, has dramatically advanced our understanding of bacterial development. Work on several well-developed model systems such as Bacillus subtilis, Caulobacter crescentus, Myxococcus xanthus and Streptomyces spp., has provided us with an in-depth understanding of processes such as sporulation, multicellular behaviour and the bacterial cell cycle. At the same time, these studies have revolutionized our view of the bacterial cell and shown it to be a highly complex entity with spatial and temporal organization. The recent American Society for Microbiology (ASM) conference on prokaryotic development demonstrated that several laboratories have now started to connect data obtained through functional genomic analysis with subcellular organization, thereby generating three-dimensional regulatory networks. This meeting report highlights new findings in the field, such as regulation of protein localization during sporulation and the cell cycle, control of cell-cell interaction and the initiation of cell division.     

Cell polarity and the control of apical growth in Streptomyces

Streptomyces cells grow by building cell wall at one pole-the hyphal tip. Although analogous to hyphal growth in fungi, this is achieved in a prokaryote, without any of the well-known eukaryotic cell polarity proteins, and it is also unique among bacterial cases of cell polarity. Further, polar growth of Streptomyces and the related mycobacteria and corynebacteria is independent of the MreB cytoskeleton and involves a number of coiled-coil proteins, including the polarity determinant DivIVA. Recent progress sheds light on targeting of DivIVA to hyphal tips and highlight protein phosphorylation in the regulation of actinobacterial growth. Furthermore, cell polarity affects not only cell envelope biogenesis in Streptomyces, but apparently also assembly of fimbriae, conjugation and migration of nucleoids.     

Fluorescence Time-lapse Imaging of the Complete S. venezuelae Life Cycle Using a Microfluidic Device

Live-cell imaging of biological processes at the single cell level has been instrumental to our current understanding of the subcellular organization of bacterial cells. However, the application of time-lapse microscopy to study the cell biological processes underpinning development in the sporulating filamentous bacteria Streptomyces has been hampered by technical difficulties. Here we present a protocol to overcome these limitations by growing the new model species, Streptomyces venezuelae, in a commercially available microfluidic device which is connected to an inverted fluorescence widefield microscope. Unlike the classical model species, Streptomyces coelicolor, S. venezuelae sporulates in liquid, allowing the application of microfluidic growth chambers to cultivate and microscopically monitor the cellular development and differentiation of S. venezuelae over long time periods. In addition to monitoring morphological changes, the spatio-temporal distribution of fluorescently labeled target proteins can also be visualized by time-lapse microscopy. Moreover, the microfluidic platform offers the experimental flexibility to exchange the culture medium, which is used in the detailed protocol to stimulate sporulation of S. venezuelae in the microfluidic chamber. Images of the entire S. venezuelae life cycle are acquired at specific intervals and processed in the open-source software Fiji to produce movies of the recorded time-series.     

Sequence similarity between macrolide-resistance determinants and ATP-binding transport proteins.

The three macrolide-resistance-encoding genes, tlrC from Streptomyces fradiae, srmB from Streptomyces ambofaciens, and carA from Streptomyces thermotolerans, encode proteins that possess significant sequence similarity to ATP-dependent transport proteins. The N-terminal and C-terminal halves of these proteins are very similar to each other and contain highly conserved regions that resemble ATP-binding domains typically present within the superfamily of ATP-dependent transport proteins. These observations suggest that the mechanism by which these genes confer resistance to macrolides is due to export of the antibiotics, a process that is driven by energy derived from ATP hydrolysis.     

TIR-containing adaptors in Toll-like receptor signalling

While the Toll-like receptors (TLRs) are responsible for the recognition and response to pathogen ligands, increasing evidence suggests that the family of five cytosolic Toll/IL-1 receptor (TIR) adaptor proteins also play a crucial role in the specificity of the response. Genetic studies in mice, and increasingly in human polymorphic populations, have given us a greater understanding the role these adaptors play in orchestrating and coordinating the multifaceted immune response to multiple exogenous threats. Importantly, with growing evidence of the critical role TLRs play in responses to host danger signals and autoimmune disease, a more comprehensive understanding and appreciation of the role these adaptors play in disease progression may provide future targets for therapeutic intervention in human disease. Importantly, growing evidence supports the concept of pathway specific and inflammatory control by a better understanding of how these adaptors interact with other signalling mediators, where they localise within the cell and the inflammatory programs they initiate as a way of manipulating immune responses. This review deals with our current understanding of these TIR-containing adaptor proteins and how mutagenesis of specific residues and domains has increased our knowledge of their function in TLR immune responses.     

Maintaining copper homeostasis: regulation of copper-trafficking proteins in response to copper deficiency or overload

Copper is an essential micronutrient that plays a vital role as a catalytic co-factor for a variety of metalloenzymes. The redox chemistry of copper also makes it a potentially toxic metal if not properly used. Therefore, elaborate mechanisms have evolved for controlling its cellular uptake, elimination, and distribution. In the last decade, our understanding of the systems involved in maintaining copper homeostasis has improved considerably with the characterization of copper transporters that mediate cellular copper uptake or efflux and with the identification of copper chaperones, a family of proteins required for delivering copper to specific targets in the cell. Despite the distinct roles of these proteins in copper trafficking, all seem able to respond to changes in copper status. Here, we describe recent advances in our knowledge of how copper-trafficking proteins respond to copper deficiency or overload in mammalian cells in order to maintain copper balance.     

New Knowledge from Old: In silico discovery of novel protein domains in Streptomyces coelicolor

Background

Streptomyces coelicolor has long been considered a remarkable bacterium with a complex life-cycle, ubiquitous environmental distribution, linear chromosomes and plasmids, and a huge range of pharmaceutically useful secondary metabolites. Completion of the genome sequence demonstrated that this diversity carried through to the genetic level, with over 7000 genes identified. We sought to expand our understanding of this organism at the molecular level through identification and annotation of novel protein domains. Protein domains are the evolutionary conserved units from which proteins are formed.

Results

Two automated methods were employed to rapidly generate an optimised set of targets, which were subsequently analysed manually. A final set of 37 domains or structural repeats, represented 204 times in the genome, was developed. Using these families enabled us to correlate items of information from many different resources. Several immediately enhance our understanding both of S. coelicolor and also general bacterial molecular mechanisms, including cell wall biosynthesis regulation and streptomycete telomere maintenance.

Discussion

Delineation of protein domain families enables detailed analysis of protein function, as well as identification of likely regions or residues of particular interest. Hence this kind of prior approach can increase the rate of discovery in the laboratory. Furthermore we demonstrate that using this type of in silico method it is possible to fairly rapidly generate new biological information from previously uncorrelated data.     

From the growth cone surface to the cytoskeleton: one journey, many paths

The mechanisms that guide axons through a complex cellular landscape to reach appropriate target cells are central to our understanding of neural development. Decades of work suggest that guidance information is interpreted by signaling machinery that controls the complex and dynamic cytoskeleton at the growth cone leading edge. Recent insights from the areas of signal transduction and cell biology have identified a number of key components that play central roles in this chain of command, including members of the Ena/VASP and WASP family of proteins. Although our understanding of the precise mechanism by which these proteins control actin assembly is still incomplete, these players are emerging as potential sites of integration that translate convergent signals into directional cell movement. This brief review explores some of the most recent articles on this topic.     

Selection of polarized growth sites in yeast

The budding yeast Saccharomyces cerevisiae responds to intracellular and extracellular cues to direct cell growth. Genetic analysis has revealed many components that participate in this process and has provided insight into the mechanisms by which these proteins function. Several of these components, such as the septins, pheromone receptors and GTPase proteins, have homologues in multicellular eukaryotes, suggesting that many aspects of polarized cell growth may be conserved throughout evolution. This review discusses our current understanding of the molecular mechanisms of growth-site selection during the different stages of the yeast life cycle.     

Monomeric red fluorescent protein as a reporter for macromolecular localization in Streptomyces coelicolor

The enhanced green fluorescent protein (eGFP) is widely used to investigate cell type specific gene expression and protein localization in the filamentous streptomycetes. To broaden the scope of cell biological investigation in these organisms, we have adapted shuttle vectors for the construction of gene fusions to the monomeric red fluorescent protein (mRFP1) and have tested them in Streptomyces coelicolor. Using fusions of mRFP1 to the cell division proteins DivIVA and FtsZ, we show that mRFP1 is comparable to eGFP for cell biological research in this organism and suggest that this paves the way for the future use of two-color imaging and FRET.     

Computational design of membrane proteins

Membrane proteins are involved in a wide variety of cellular processes, and are typically part of the first interaction a cell has with extracellular molecules. As a result, these proteins comprise a majority of known drug targets. Membrane proteins are among the most difficult proteins to obtain and characterize, and a structure-based understanding of their properties can be difficult to elucidate. Notwithstanding, the design of membrane proteins can provide stringent tests of our understanding of these crucial biological systems, as well as introduce novel or targeted functionalities. Computational design methods have been particularly helpful in addressing these issues, and this review discusses recent studies that tailor membrane proteins to display specific structures or functions and examines how redesigned membrane proteins are being used to facilitate structural and functional studies.     

Taking the Scenic Route: Biosynthetic Traffic to the Plasma Membrane in Polarized Epithelial Cells

The maintenance of epithelial cell function requires the establishment and continuous renewal of differentiated apical and basolateral plasma membrane domains with distinct lipid and protein compositions. Newly synthesized proteins destined for either surface domain are processed along the biosynthetic pathway and segregated into distinct subsets of transport carriers emanating from the trans -Golgi network. Recent studies have illuminated additional complexities in the subsequent delivery of these proteins to the cell surface. In particular, multiple routes to the apical and basolateral cell surfaces have been uncovered, and many of these involve indirect passage through endocytic compartments. This review summarizes our current understanding of these routes and discusses open issues that remain to be clarified.